Optimized Time-Segmented Acquisition Expands Peptide and Protein Identification in TIMS-TOF Pro Mass Spectrometry

Abstract

We introduce here a novel approach, termed time-segmented acquisition (Seg), to enhance the identification of peptides and proteins in trapped ion mobility spectrometry (TIMS)-time-of-flight (TOF) mass spectrometry. Our method exploits the positive correlation between ion mobility values and reversed-phase liquid chromatography (LC) retention time to improve ion separation and resolution. By dividing the LC retention time into multiple segments and applying a segment-specific narrower ion mobility range within the TIMS tunnel, we achieved better separation and higher resolution of ion mobility. In comparison to conventional TIMS methods, which typically scan a static ion mobility range (either from 0.6 to 1.6 [Wide] or from 0.85 to 1.3 [Narrow], V × s/cm²), the Seg method demonstrates marked improvements in identification rates. Compared to Wide scanning, the Seg method increases peptide identifications by 17-27% and protein identifications by 6-16% depending on the gradient length and the sample load. The enhancement in peptide identification is even more pronounced when compared to Narrow scanning, with an increase of 34-86%. These findings highlight the potential of the Seg dda-PASEF method in expanding the capabilities of TIMS-TOF mass spectrometry, especially for peptide-focused analyses, such as post-translational modifications and peptidomics.

Keywords: TIMS-TOF; dda-PASEF; ion mobility; label-free quantitation; phosphoproteomics.

Figure 1

Temporal peptide ion mobility profiles over the 30- (A, B), 60- (C, D), and 90 min (E, F) LC-gradients. Peptide samples were analyzed using a conventional PASEF method with a wide range of ion mobility (0.60 to 1.60 V·s/cm²). The “s” denotes sample loading amount used in each experiment. Majority of peptides were found within the two narrow-ranged (of ion mobility) red dashed lines. The color bar indicates percentage of peptides from overall identification. The red lines define the boundaries of the identified peptides in each experiment condition.

Figure 2

Design of the time-segmented methods for three LC-gradients (A, 30 min; B, 60 min; C, 90 min) according to their ion elution profiles. A total of 9–11 segments, each having a narrower ion mobility scan window compared to the commonly used ion mobility 1/K₀ range of 0.6–1.6 V·s/cm². Sample loading amount (s) and LC retention time (RT) are indicated in each experiment.

Figure 3

Numbers (mean ± standard error) of proteins (A), peptides (B), and average number of peptides per identified protein (C) identified from 5–200 ng triplicate injections of HeLa digest using the three TIMS scan acquisition methods: the Wide, Narrow, and Seg method.

Figure 4

Protein and peptide analysis of HeLa digest (200 ng) using three MS-acquisition methods. (A, B) Unweighted Venn diagrams were used for better visualization of the unique and overlap of identified proteins (A) and peptides (B). (C–E) Distribution of total (colored) and unique (black) peptide abundance in Wide (C, blue), Narrow (D, red), and Seg (E, green) methods. Natural log-transformed intensities plotted against frequency.

Figure 5

Reproducibility and correlation analysis of protein quantification across methods. (A, B) Correlation of natural log-transformed protein intensities between replicates for Wide (A) and Seg (B) methods. (C, D) Coefficient of variation (CV) of protein quantities across triplicates for Wide (C) and Seg (D) methods. (E) Correlation of natural log-transformed protein intensities between Wide and Seg methods. The r denotes the Pearson correlation coefficients.

Figure 6

Comparison of the Wide and Seg methods for the phosphoproteome analysis of cultured HeLa cells. (A) Distribution of identified phosphosites across HpH fractions, demonstrating enhanced phosphosite identification by the Seg method. (B) Comparative analysis of modified peptides identified by each method, with the Seg method identifying twice as many unique peptides as the Wide method. (C) Venn diagram illustrating the overlap and unique identification of phosphosites between the two methods. (D) Total number of identified phosphosites from tryptic and semitryptic database searches, respectively. Quantitative comparison of natural log-transformed intensities of identified phosphopeptides (E) and phosphoproteins (F) between Wide and Seg, demonstrating a high correlation coefficient (r) and strong agreement in quantification between the two methods.

Figure 7

TIMS resolution of three representative peptide ions and identified GAPDH peptides. (A) Representative LC-MS spectra showing base peak chromogram, extracted ion chromograms (EIC) for three “house-keeping” ions, and LC running gradient (solvent B composition) of 200 ng of HeLa digest. (B, C) Comparative bar graph of extracted ion mobility (EIM) showing the differences in TIMS resolution (mean ± standard error of triplicate injections) of the three ions (B) and the commonly identified GAPDH peptides (C) from the Wide, Narrow and Seg methods.